The most important parameters for the bone architecture are the forces’ direction, intensity and cyclicity.
The bone can sense them and discriminate with different devices, those elements modulate the bone remodelling to create the best bone configuration. The mechanisms behind this remodelling are still not well known, the studies go through different ways but there are two most diffused theory:
- One is based on osteocytes as mechanosensors, those cells have particular parts that can detect the changes of fluid-flow in the osteocyte network(for the mechanical loadings) adapting the bone structure;
- The second idea is that fluid itself could be ionically charged and its movement(for strain or distension) creates a ionic flow transformed in a chemical signal by the osteocyte, this theory is now almost abandoned.
Osteocyte as mechanosensory cells
This theory is diffused because of the osteocyte peculiar position in the matrix, they are inserted in cavity called lacunes where spread through canaliculies their cytoplasmic prolongaments. The canaliculies create a network among the osteocytes that can communicate and carry out metabolic exchanges whitin them or the circulatory system(thanks to Gap Junction at the tip of dendrites).
Although we know the structure of osteocytes and its costituents it’s still not clear what are the elements that transpose the stimulus and how they render it a cellular reply. There are a lot studies each with different direction, but three are the most studied.
The idea and the evidence
The first to observe its presence in the osteocyte were Tonna and Lampen in 1972, they postulate it could be a potential sensor for mechanical stimulus for the bone.
Recent studies attribute it this feature, in fact, mechanosensory proteins( PKD1 were discovered first in cilium of kidney cells and also in osteocytes on the cilium) associated with calcium channels switch on their opening due to movement of the canalicular fluid(created by mechanical loading) leading to cascade of responses.
Calcium ion and IP3
This mechanism was observed in kidney cells, the osteocytes are a little different because they don’t take the external calcium but release the reserves increasing their calcium levels.
The calcium ion spreads in the cell activating different systems inside the cell body which leads to expression of differents genes, but it can’t go through the gap junctions which connect the cell with each other(indeed it close them), so the signal is passed with a molecule, the IP3. This molecule could cross the gap junctions and inside the cells stimulate the release of calcium reserves, its synthesis is regulated by the level of cytoplasmic calcium(the cascade activated by the high level calcium initiate the production of IP3).
The increase of calcium and The PDK1, for changes due to its stretching, lead to the synhesis of a factor(transcription factor Runx2) which stimulates the osteoblasts proliferation and differentiation to maintain or strengthen bone status. Studies demonstrate loss of cilia lead to a lower fluid-flow sensitivity and a weaker bone. These cilia could pick up the external mechanical stimuli, discriminate according to the intensity, the duration, the orientation and adapting the cellular response.
Model diagram for the role of hemichannels under fluid flow shear stress in osteocytes. Hemichannels are expressed on the plasma membrane away from cell-cell junction regions. (A) In the absence of mechanical stress, hemichannels remain closed, whereas gap junctions are kept open. (B) Fluid flow shear stress induces the opening of hemichannels (upper panel). PGE2, possibly ATP and other responding physiological factors are released into canaliculi to mediate biological responses elicited by mechanical stress (lower panel).
The fluid-flow and sensations
The osteocyte has some cytoplasmatic prolongaments called dendrites, the hypothesis is these elements can detect the canalicular flow and reply with the transmission of the signal to the cell body.
Some experiments have demonstrated the flow was perceived through some filaments that tethered the dendrites to the canalicular wall, but this doesn't explain which is the receptive zone. To explain the mechanism of action we need to know some molecules called connexons, these molecules are important elements in the formation of gap junction and hemichannels which let the molecular transport of the cell trough the matrix.
The dendrites sensibility
Studies on cultured osteocyte demonstrate the hemichannels opening depends on the area of the stimulus (the dendritic area is receptive but less responsive) but in general the fluid flow activates this channels and this leads to the release of PGE2 and NO(important elements for the bone vitality, the first is a stimulator of bone resorption and the seod one for the osteocyte activation) in the cell body. Mechanical stimuli lead to an opening in the cell body and less in the dendrites, because their function is to detect and transmit the signal to the cell body that responds.
The dendrites can detect the changes in the fluid but to improve the sensitivity the cells create a binding with the canalicular wall trough the glycocalyx, there are molecules called integrins on it that bind the wall so that changes in the fluid not so great could be detected better, damages to this structure leads to a big loss of hemichannels opening on the cell body(the cell body can also locate the stimuli but with more less sensibility).
The conclusion is the osteocyte can detect the stimuli trough the dendrites and its support structures, transmit the signal to the cell body that can reply with the bone remodelling. The studies were conducted with systems “in vitro”, so it’s still not sure how the ostecytes can react in the body, but for sure this pathway can be a possibility.
Histology (A–C; original magnification, ×40) and composite schematic (D) of the BRC, which comprises the cells constituting the BMU — specifically osteoclasts (OCs), osteoblasts (OBs), and osteocytes — as well as the canopy of bone-lining cells and the associated capillary.
The osteocyte location
The osteocyte is locked in a particular location called lacuna but it can always communicate with the others one trough its prolongaments in the canaliculies. The stimulation and communication for the osteocyte is vital, the absence of either leads to the cellular apoptosis. The mechanical stimuli change the flow of the canalicular fluid and this helps the movement of nutrients and oxygen, this is better of the mere diffusion because the elements arrive also in the areas more distant from the vessels. The lost of loading means the absence of flow and the state of hypoxia in the emarginated cells, this activate the apoptosis program and the release of factors like osteopontin(molecules that incite the osteoclasts bone resorption).
When the bone is subjected to unexpected stimuli the intercommunication structure can be damaged and the interactions lost. This is the example of microcrack in the bone tissue, the destruction of the compact matrix leads to apoptosis of the closest osteocytes.
During normal activity the osteocyte releases the sclerostin, this molecule is a inhibitor of the Wnt signalling in the cells near the surface. The osteocytes still alive can detect the microdamage and start to release growth factor, prostaglandins and nitric oxide. In the meanwhile the lining cells pull away from the bone matrix and form a canopy which merges with the blood vessels. The apoptosis of osteocytes activates also the production of TRACP by the osteoclasts, this means a relationship between them and osteocyte(similar to the osteoblats).
The arrival of osteoclasts
With the death of the osteocytes the stromal cells are no more inhibited and start to express Wnt and other factors(for example the interlukin-1 that stimulates the proliferation), the lost of the inhibition leads to a proliferation and differentiation into pre-osteoblasts and the release of M-CSF (macrophage colony-stimulating factor, which helps the staminal cells to generate pre-osteoclasts). The pre-osteoblasts also release some factors for the self-stimulation and proliferation such as Wnt, interleukins and bone morphogenic proteins.
The free osteclasts in the bone bloodstream are attracted in the microcrack zone with the canopy, stem cells near the vessels wall differentiate in pre-osteoclasts to increase their number, and on their surface express a receptor for a pre-osteoblasts molecule which leads to their activation. The pre-osteoblasts in their differentiation start to express on the surface RANK-L, this molecule is the target of pre-osteocalsts receptor that leads to their activation, the binding activates an enlargement process first and then the cells fusion forming mature osteoclasts.
Recognition and resorption of the area
With the osteoclasts activation starts the bone resorption, the osteoclasts bind to the compact matrix with the integrins and stars to secrete acids and cathepsin K. This process is very long, it can go on for a couple of week, to stop it bone-derived growth factors IGF (osteocyte activator) and TGF-β(osteoclast inhibitor) are released. At the end of the job the osteoclasts undergoes apoptosis, their lifespan is regulated by estrogen and other factors.
The osteoclasts can detect the microcrack area thanks to some molecules in the canalicular fluid, the apoptosis of osteocyte leads to a release of its molecules, some of the most present on the membrane are RANK-L and M-CSF that activate the TRACP production in the osteoclasts and their osteoclastic activity. The osteocytes normally release TGF-β to inhibit the osteoclasts, but with the apoptosis the levels of M-CSF and RANK-L are increased and the action of TGF-β is silenced.
Now is time to reconstruct the matrix so the pre-osteoblasts mature and become osteoblasts(they end to express RANK-L), they start to secret OPG(osteoprotegerin) that bind RANK-L stopping the pre-osteoclasts activation and meantime they’re proliferating and aligning the resorbed cavity. This osteoblasts start to secret the matrix(osteoid) and go on till the cavity is full, they release also growth factors like IGF and TGF-β. In the process of redeposit some osteblasts will be leaved in the matrix and are going to be osteocyte, others will be lining cells and the leftovers will undergo apoptosis.
Now the matrix is completely set the canopy could disappear dividing from the bloodstream, however the matrix is still not mineralized in this way the osteocytes can make prolongaments to communicate with the neighboring. For the complete remodelling the bone needs about three years where the matrix stores minerals and increases its density.
The complex of cells that worked in this process is called BMU, this is the starting point for the remodelling.